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Item The ability of new intracanal medicaments to prevent the formation of multi-species biofilm on radicular dentin(2017) Jacobs, Jordon C.; Spolnik, Kenneth J.; Ehrlich, Ygal; Gregory, Richard L.; Yassen, Ghaeth; Bringas, JosefThe residual antibacterial effects of antimicrobials used in endodontic regeneration against biofilm bacteria obtained from immature and mature teeth Jordon C. Jacobs DDS, Richard L Gregory PhD, Ygal Ehrlich DMD, Kenneth Spolnik DDS, MS, Josef S. Bringas DMD, DDS, MS, and Ghaeth Yassen BDS, MSD, PhD We explored the residual antibacterial properties of dentin pretreated with low concentrations of double antibiotic paste (DAP) against biofilm bacteria obtained from different clinical sources. Dentin blocks were sterilized and randomized into 4 treatment groups and 2 control groups (n=20). Blocks from treatment groups were pretreated with DAP (1 or 5 mg/ml) loaded into methylcellulose, calcium hydroxide (Ca(OH)2), or methylcellulose paste. After one week, the treatment pastes were removed and all blocks were immersed in PBS. The dentin blocks from treatment groups and one of the control groups were then inoculated with bacterial isolates obtained from immature or mature teeth with pulpal necrosis(n=10). The remaining control group received no bacteria and was used as a sterile control. Blocks were then incubated anaerobically for 3 weeks. Biofilm disruption assays were conducted for all samples. Two-way ANOVA and pair-wise comparisons were used for statistical analyses. The residual antibacterial effect of dentin pretreated with 5 mg/ml of DAP was significantly higher than all other groups regardless of the source of biofilm. Dentin pretreated with 1 mg/ml of DAP demonstrated significantly higher residual antibacterial effects in comparison to dentin pretreated with placebo paste and Ca(OH)2 only in bacterial isolates obtained from mature teeth with pulpal necrosis. Dentin pretreated with Ca(OH)2 did not demonstrate any residual antibacterial effects. Dentin pretreated with 1 or 5 mg/ml of DAP demonstrated significantly better residual antibacterial effects against biofilm bacteria obtained from mature teeth with pulpal necrosis in comparison to bacterial isolates obtained from immature teeth with pulpal necrosis.Item The antibacterial effect of a radiopaque double antibiotic paste against both an established multispecies and a single enterococcus faecalis biofilm(2019) Haslam, Bryce S.; Spolnik, Kenneth J.; Ehrlich, Ygal; Gregory, Richard L.; Bringas, Josef; Warner, Ned A.For regenerative endodontic procedures (REPs) to be successful an elimination of bacteria from the root canal system must be accomplished. Many different medicaments with antibacterial properties have been used to obtain complete disinfection. Double antibiotic paste (DAP) containing a mixture of ciprofloxacin and metronidazole has been shown to be a promising intracanal medicament. The addition of a radiopaque filler such as zirconium oxide to DAP may affect the antibacterial properties of DAP as well as allow precise placement and radiographic visualization of its position in the canal system. The aim of the proposed study was to evaluate the direct antibacterial properties of zirconium oxide radiopacifier combined with DAP (RoDAP) against a multispecies biofilm from a bacterial isolate from an infected immature tooth with a necrotic pulp and a known single species biofilm. 4x4 mm radicular dentin specimens (n = 112) obtained from human extracted teeth were used prepared and sterilized prior to use. A multispecies clinical bacterial isolate from an immature tooth with a necrotic pulp and a single species Enterococcus faecalis isolate were obtained. These bacterial isolates were used to inoculate dentin slabs and grown for 3 weeks. The dentin slabs were treated for 1 week with 1.0-mg/mL and 10- mg/mL RoDAP, 1.0-mg/mL DAP, and two placebo pastes consisting of methyl cellulose (MC) and methyl cellulose combined with zirconium oxide (RoMC), respectively, as well as two no-treatment controls. Following treatment, the grown biofilm was detached and spiral plated. The plated biofilm cells were cultured for 24 hours and each group examined using a colony counter to determine bacterial numbers (CFUs/mL). Data analysis, using a 5.0-percent significance level was conducted using one-way ANOVA followed by pair-wise group comparisons. Both 1.0-mg/mL and 10 mg/mL RoDAP demonstrated significant antibacterial effects against bacterial isolates from an immature tooth with a necrotic pulp as well as an E. faecalis isolate. The precise application of RoDAP confirmed radiographically with its direct antibacterial properties may be beneficial for intracanal disinfection during REPs.Item The antibacterial effect of new intracanal medicaments against established mutlispecies biofilm(2017) Troxel, Alex; Spolnik, Kenneth J.; Gregory, Richard; Ehrlich, Ygal; Bringas, Josef; Zunt, Susan L.; Yassen, GhaethWe investigated the antibacterial effect of low concentrations of double antibiotic paste (DAP) loaded into a methylcellulose system against bacterial biofilms obtained from mature and immature teeth with necrotic pulps. Standardized radicular dentin specimens were randomly divided into six experimental groups (n = 20). Group 1: 5mg/mL DAP treatment. Group 2: 1mg/mL DAP treatment. Group 3: Calcium hydroxide (Ca(OH)2) treatment. Group 4: Methylcellulose. Group 5: No treatment. Group 6: No bacteria or treatment. Clinical bacterial isolates were obtained from mature and immature teeth with necrotic pulps indicated for endodontic regeneration or routine endodontic treatment, respectively. Specimens in each group were inoculated with either bacterial isolates (n = 10) and incubated anaerobically for 3 weeks. Specimens were then treated for one week with the assigned group treatment. Treatments were rinsed with sterile saline and biofilms were detached and spiral plated using biofilm disruption assays. Wilcoxon Rank Sum tests followed by pair-wise comparisons were used for statistical analyses. Treatment of infected dentin with 1 mg/ml of DAP, 5 mg/mL of DAP, and Ca(OH)2 demonstrated significant and substantial antibiofilm effects in comparison to untreated control groups or groups treated with placebo paste. Furthermore, 1 mg/mL of DAP caused complete eradication of biofilm obtained from mature tooth with necrotic pulp. However, the same concentration was not able to completely eradicate biofilm obtained from the immature tooth with necrotic pulp. Low concentrations of DAP (1-5 mg/mL) loaded into a biocompatible methylcellulose system demonstrated significant antibacterial effects against biofilm obtained from both mature and immature teeth with necrotic pulps.Item The antibacterial effects of radiopaque double antibiotic pastes against clinical bacterial isolates from mature and immature teeth with necrotic pulps(2018) Ibrahim, Carolin Francis; Spolnik, Kenneth J.; Ehrlich, Ygal; Gregory, Richard L.; Zunt, Susan; Bringas, Josef; Yassen, GhaethLow concentrations (1-10mg/mL) of double antibiotic paste (DAP) have demonstrated antibacterial properties in regenerative endodontics. The aim of this study was to evaluate if DAP made radiopaque (RoDAP) with barium sulfate has antibacterial effects against bacterial isolates from a mature and immature tooth with necrotic pulp. Clinical bacterial isolates were obtained from the canals of mature and immature teeth with necrotic pulps during root canal therapy or a regenerative procedure, respectively. Bacterial isolates were grown anaerobically for three weeks on 4x4mm dentin specimens prepared from extracted human teeth (n=48 per biofilm type). The dentin specimens were allocated into six groups and treated as follows: 1mg/mL RoDAP, 10mg/mL RoDAP, calcium hydroxide (UltraCal), placebo (barium sulfate in methylcellulose), no treatment, and no bacteria or treatment (sterile control). After one week of treatment the biofilm was detached and biofilm disruption assays were conducted to determine the bacterial numbers (CFUs/mL). The data was analyzed using Wilcoxon Rank Sum tests followed by pairwise comparisons. 1 and 10 mg/mL RoDAP as well as calcium hydroxide demonstrated significant antibacterial effects against the tested bacterial isolates. The placebo paste did not demonstrate any significant antibacterial effects. No significant difference in antibacterial effects was found against isolates from both mature and immature teeth regardless of the type of treatment. Both 1 and 10 mg/mL RoDAP demonstrated significant antibacterial effects against bacterial isolates from mature and immature teeth with necrotic pulps. RoDAP can be beneficial clinically since its adequate placement within the canal system can be confirmed radiographically.Item The antibacterial stability of a new radiopaque double antibiotic paste(2018-06) Epkey, Kathryn Eileen; Spolnik, Kenneth J.; Ehrlich, Ygal; Gregory, Richard L.; Zunt, Susan; Bringas, Josef; Yassen, GhaethWe evaluated the antibacterial stability (shelf life) of a new radiopaque double antibiotic paste (RoDAP) loaded in a methylcellulose system with 30% w/v barium sulfate against biofilm collected from an immature tooth with necrotic pulp. Uniform radicular dentin specimens were infected with bacterial biofilm obtained from an immature tooth with a necrotic pulp and incubated anaerobically for three weeks. These samples were randomly divided into 6 experimental groups (n=7) and treated for 1 week at three time points of aged radiopaque DAP: 0 months, 3 months, and 6 months. Group 1: 1mg/mL RoDAP treatment. Group 2: 10 mg/mL RoDAP treatment. Group 3: Calcium hydroxide (Ca(OH)2) treatment. Group 4: Methylcellulose with barium sulfate. Group 5: No treatment. Group 6: No bacteria or treatment. The samples were rinsed with sterile saline to detach biofilms and then spiral plated using a biofilm disruption assay. Statistical analyses were performed using Wilcoxon rank-sum tests and Wilcoxon signed rank tests with fixed effects for treatment, time, and the treatment-by-time interaction. Treatment of infected dentin with 1 mg/mL RoDAP, 10 mg/mL RoDAP, and Ca(OH)2 demonstrated significant and substantial antibiofilm effects in comparison to untreated control groups or groups treated with placebo paste after 0, 3, and 6 months of aging. Calcium hydroxide, however, showed slightly less antibiofilm activity after 6 months of aging when compared to 0 months and 3 months of aging. This difference was statistically significant (p > 0.05). In conclusion, both concentrations of RoDAP maintained full antibacterial efficacy after 6 months of aging, while calcium hydroxide lost some antibacterial activity after a shelf life of 6 months.Item Antibacterial TAP-mimic electrospun polymer scaffold: effects on P. gingivalis-infected dentin biofilm(Springer, 2016-03) Albuquerque, Maria Tereza P.; Evans, Joshua D.; Gregory, Richard L.; Valera, Marcia C.; Bottino, Marco C.; Department of Anatomy & Cell Biology, IU School of MedicineOBJECTIVES: This study sought to investigate, in vitro, the effects of a recently developed triple antibiotic paste (TAP)-mimic polymer nanofibrous scaffold against Porphyromonas gingivalis-infected dentin biofilm. MATERIALS AND METHODS: Dentin specimens (4 × 4 × 1 mm(3)) were prepared from human canines. The specimens were sterilized, inoculated with P. gingivalis (ATCC 33277), and incubated for 1 week to allow for biofilm formation. Infected dentin specimens were exposed for 3 days to the following treatments: antibiotic-free polydioxanone scaffold (PDS, control), PDS + 25 wt% TAP [25 mg of each antibiotic (metronidazole, ciprofloxacin, and minocycline) per mL of the PDS polymer solution], or a saturated TAP-based solution (50 mg of each antibiotic per mL of saline solution). In order to serve as the negative control, infected dentin specimens were left untreated (bacteria only). To determine the antimicrobial efficacy of the TAP-mimic scaffold, a colony-forming unit (CFU) per milliliter (n = 10/group) measurement was performed. Furthermore, additional specimens (n = 2/group) were prepared to qualitatively study biofilm inhibition via scanning electron microscopy (SEM). Statistics were performed, and significance was set at the 5% level. RESULTS: Both the TAP-mimic scaffold and the positive control (TAP solution) led to complete bacterial elimination, differing statistically (p < 0.05) from the negative control group (bacteria only). No statistical differences were observed for CFU per milliliter data between antibiotic-free scaffolds (2.7 log10 CFU/mL) and the negative control (5.9 log10 CFU/mL). CONCLUSIONS: The obtained data revealed significant antimicrobial properties of the novel PDS-based TAP-mimic scaffold against an established P. gingivalis-infected dentin biofilm. CLINICAL RELEVANCE: Collectively, the data suggest that the proposed nanofibrous scaffold might be used as an alternative to the advocated clinical gold standard (i.e., TAP) for intracanal disinfection prior to regenerative endodontics.Item Bactericidal Efficacy of EdgePRO Er,Cr:YSGG Laser-Activated Irrigation Against a Mature Endodontic Multispecies Biofilm Using an in vitro Infected Tooth Model(2024) Patterson, Samuel B.; Spolnik, Kenneth J.; Gregory, Richard; Ehrlich, Ygal; Movila, AlexandruIntroduction: Treatment goals of non-surgical root canal therapy (nsRCT) include the removal of all organic tissue material, bacterial biofilm and their by-products, and debris materials, in order to disinfect the canal system to a level compatible with healing and to further prevent infection. Standard chemo-mechanical protocols have several well-documented shortcomings and subsequent areas for improvement regarding their disinfection abilities. In recent years, emerging laser technology and its application in root canal therapy has been gaining popularity as a safe and promising tool for advancing endodontic treatment. The newest FDA-approved laser for endodontic application is the EdgePRO Erbium,Chromium-doped:Yttrium-Scandium-Gallium-Garnet (Er,Cr:YSGG) infrared laser operating at a 2780 nm wavelength. Previous in vitro studies using Er,Cr:YSGG lasers have demonstrated their ability to enhanced canal debridement, cleaning, smear layer removal, and bacterial disinfection. Additionally, a few in vivo trails have been completed using this laser type as an adjunct in RCT procedures, which have yielded safe and highly successful results in the clinical setting. However, research specifically using the EdgePro device as well as a standardized protocol for optimal clinical usage of the laser is lacking. Objectives: The aim of this study was to evaluate the bactericidal and biofilm dissolution effects of laser-activated irrigation using the EdgePro laser against a mature multispecies biofilm in an infected tooth model and to assess the potential increased disinfection and cleaning ability compared to a standard needle irrigation protocol. Materials and Methods: Single rooted teeth (n=36) were decoronated to a standardized length of 16mm. The root canals were endodontically prepared using a standard irrigation, hand-filing, and rotary protocol to a final size of ISO 25.06 while maintaining a fully patent apical foramen. An irrigation solution reservoir was created in the coronal 4 mm of the canal space. Sterile specimens were inoculated with multispecies bacterial sample containing E. faecalis. The mixed bacteria was grown anaerobically for 10 days to form a mature biofilm using a previously established protocol. The teeth were divided into a negative control group (saline rinse, n=12), positive control group (standard needle irrigation – SNI, n=12), and an experimental group (laser-assisted treatment protocol, n=12). The positive control and experimental laser groups utilized the same irrigation solutions of 2 mL 17% EDTA followed by 5 mL 3% NaOCl using a standard 27-gauge side-vented irrigation needle placed as far apically as possible without binding. The experimental group underwent additional laser activation using laser tip #2 (350 m diameter) and settings of: 15 mJ, 0.75 W, 50 Hz, 0% air, and 0% water spray (Mid-Root Solutions 1 preset). The laser tip was inserted halfway into the irrigation filled canals (8 mm from orifice and apex) and fired upon withdrawal at a speed of 0.8 mm/sec, which comprised a single lasing cycle of 10 seconds. Three lasing cycles were completed with EDTA first followed by NaOCl, for a total of six lasing cycles with 60 seconds of irradiation time per tooth. A final rinse of sterile saline was used in all tooth samples prior to bacterial sample collection via Versa-brushes and sterile paper points. The samples were transferred to a laboratory setting where they underwent ultrasonic agitation, serial dilution, spiral plating on blood-agar, and two days of anaerobic incubation for assessment of bacterial growth. Colony forming units (CFUs/mL) were counted as a means of quantitative analysis. Results: The negative control group yielded the highest level of bacterial growth with an average of 934,771 CFUs/mL. The positive control group displayed a statistically significant lower amount of bacterial growth with an average of 4,698 CFUs/mL and yielded 1 sample with no bacterial growth. The experimental laser group had statistically significant lower bacterial growth present compared to both the positive and negative control groups and produced all negative bacterial samples with none of the 12 agar plates demonstrating CFU growth and averaged 0 CFUs/mL.. Conclusion: Within the scope of this study, laser-activated irrigation (LAI) using the EdgePro Er,Cr:YSGG laser was capable of producing no detectable bacterial samples in an in vitro infected tooth model. EdgePro LAI displayed statistically significant superior cleaning and disinfection of infected canal space compared to teeth treated with standard needle irrigation alone. The EdgePro laser system indeed shows promise as an adjunctive tool in clinical root canal treatment procedures. Further investigation is warranted using similar protocols in teeth with more complicated anatomy and with supplemental methods for analyzing bactericidal potential.Item Comparing Brief Relaxation Period to Virtual Reality Period in Reducing Dental Anxiety Prior to Root Canal Treatment: A Randomized Control Trial(2023-06) Mintz, Caley Faith; Spolnik, Kenneth; Ehrlich, Ygal; Schrader, Stuart; Flowers, Leslie; Warner, NedIntroduction: Anxiety is a debilitating and difficult sensation many people face on a daily basis. Up to 20% of American adults experience dental anxiety2. Dental anxiety can present both psychologically and physiologically as a barrier to starting, completing and/or finishing dental treatment.2,4 Catastrophizing the procedure, which is common practice in anxious patients, will alter and increase the perception of pain66. Approaching and understanding dental anxiety has shown to produce more positive treatment outcomes and overall increased patient satisfaction2, 4, 6. Non-pharmacological approaches to reducing dental anxiety can be a quick, non-invasive, method to put patients at ease and can save chair time for both practitioner and patient, as well as a more pleasant experience for the patient. Objectives: The goal of this study is to investigate non-pharmacologic approaches to reducing dental anxiety prior to non-surgical root canal treatment. This will be done by comparing an Auditory Alone Brief Relaxation period (ABR) to a Relaxation Virtual Reality period (RVR). Materials and Methods: 60 subjects who need non-surgical root canal treatment was randomly allocated into 2 groups. One group received earphones to listen to a guided brief relaxation recording, focusing on breathing and a body scan. The other group received virtual reality goggles and choose a scene of their liking to experience. State Trait Anxiety Indicator (STAI), Visual Analog Scale (VAS), and vitals were measured to objectively determine baseline anxiety score followed by the same metrics after brief relaxation or virtual reality experience. A student will perform root canal treatment, and VAS and vitals were again be recorded at the end of the appointment. Results: Both RVR and ABR showed a statistically significant decrease in anxiety in STAI-S (p value <.001 for both), STAI-T (p value 0.025 ABR; <.001 RVR). VAS scores also significantly reduced from T1 to T2 to T3 time frame. Discussion: Our study investigated and compared two different interventions in reducing anxiety prior to endodontic therapy. The results showed that both ABR and RVR reduced anxiety prior to endodontic therapy and had an effect even after the endodontic therapy was completed. Additionally, when comparing the time points T1, T2, and T3, heart rate decreased throughout the procedure and more importantly, after the intervention (ABR or RVR) was delivered. Both anxiety scales, STAI and VAS showed a statistical significant decrease in anxiety throughout the appointment. Conclusion: Non-pharmacological techniques like guided meditation and virtual reality are a valid and unique approach to reducing anxiety prior to endodontic therapy. Both ABR and RVR interventions reduced the feelings of anxiety throughout the entire endodontic appointment. This study displays the ease of incorporating both interventions to reduce anxiety in an economical and noninvasive fashion.Item A comparison of the formocresol pulpotomy technique with the calcium hydroxide pulpotomy technique(1961) Doyle, Walter A.; Mitchell, David F.Item Continued apical development of pulpless permanent teeth following endontic therapy(1968) Camp, Joe Henderson, 1939-; Van Huysen, Grant; Garner, La Forrest Dean, 1933-; Norman, Richard Daviess, 1927-; Gish, Charles W., 1923-; McDonald, Ralph E., 1920-This was a histologic study of continued apical development in the pulpless permanent teeth of dogs. The vital pulps were exposed to the oral fluids for one week. The root canals were filled with either calcium hydroxide and camphorated parachlorophenol (CMCP) or calcium hydroxide and distilled water. A vital dye, Procion red was injected to demonstrate the formation of calcified tissue. After four months, the animals were sacrificed. Decalcified semi-serial sections were studied. In 31 of 61 pulpless permanent teeth, with incompletely developed roots, continued apical development occurred. Apical calcified tissue resembling cementum was observed in 55 per cent of the specimens treated with calcium hydroxide and CMCP and in 42 per cent of those, with calcium hydroxide and distilled water. Complete closure of the apical foramen was observed in 29 and 11 per cent of the specimens respectively. Inflammation of the periapical tissues was present in 48 per cent of the calcium hydroxide and CMCP group and in 79 per cent of the other group. A significant association was found between the degree of inflammation and apical closure, (P<.001) for the calcium hydroxide-CMCP group, (P<.005) for the calcium hydroxide-distilled water group. The results suggest that calcium hydroxide and CMCP was superior to calcium hydroxide and distilled water and that the addition of CMCP to the paste reduced inflammation. Procion dye was shown to be an effective in vivo dental hard tissue marker.
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